Biomedical subjects

Takayoshi Kirisako

Publications and source records attributed to Takayoshi Kirisako.

4 recordsLinked to original sources

PubMed2006

A ubiquitin ligase complex assembles linear polyubiquitin chains.

The ubiquitin system plays important roles in the regulation of numerous cellular processes by conjugating ubiquitin to target proteins. In most cases, conjugation of polyubiquitin to target proteins regulates their function. In the polyubiquitin chains reported to date, ubiquitin monomers are linked via isopeptide bonds between an internal Lys and a C-terminal Gly. Here, we report that a protein complex consisting of two RING finger proteins, HOIL-1L and HOIP, exhibits ubiquitin polymerization activity by recognizing ubiquitin moieties of proteins. The polyubiquitin chain generated by the complex is not formed by Lys linkages, but by linkages between the C- and N-termini of ubiquitin, indicating that the ligase complex possesses a unique feature to assemble a novel head-to-tail linear polyubiquitin chain. Moreover, the complex regulates the stability of Ub-GFP (a GFP fusion protein with an N-terminal ubiquitin). The linear polyubiquitin chain generated post-translationally may function as a new modulator of proteins.

HeLa Cells↗

PubMed2005

Involvement of heme regulatory motif in heme-mediated ubiquitination and degradation of IRP2.

Iron regulatory protein 2 (IRP2), a regulator of iron metabolism, is modulated by ubiquitination and degradation. We have shown that IRP2 degradation is triggered by heme-mediated oxidation. We report here that not only Cys201, an invariant residue in the heme regulatory motif (HRM), but also His204 is critical for IRP2 degradation. Spectroscopic studies revealed that Cys201 binds ferric heme, whereas His204 is a ferrous heme binding site, indicating the involvement of these residues in sensing the redox state of the heme iron and in generating the oxidative modification. Moreover, the HRM in IRP2 has been suggested to play a critical role in its recognition by the HOIL-1 ubiquitin ligase. Although HRMs are known to sense heme concentration by simply binding to heme, the HRM in IRP2 specifically contributes to its oxidative modification, its recognition by the ligase, and its sensing of iron concentration after iron is integrated into heme.

Amino Acid Motifs↗

PubMed2005

Identification, expression, and assay of an oxidation-specific ubiquitin ligase, HOIL-1.

The ubiquitin system plays important roles in the regulation of numerous cellular processes. It is well established that ubiquitin ligases (E3s) are key components in determining the specificity of the system and that the modification of substrates such as phosphorylation often plays a critical role in selective substrate recognition by E3s. Through studies analyzing iron-mediated degradation of iron regulatory protein 2 (IRP2), a central regulator of iron metabolism in mammalian cells, we have identified a RING finger protein, HOIL-1, as an ubiquitin ligase recognizing IRP2 through a signal created by heme-mediated oxidative modification of the protein. We have utilized several types of in vitro ubiquitination assays that detect IRP2 ubiquitination and a differential yeast two-hybrid screen in which yeast cells were cultured either in the presence or in the absence of oxygen to control the oxidation state of the bait in the cells in our studies. This chapter describes the detailed methods used for the identification and functional analysis of the HOIL-1 ligase.

Animals↗

PubMed2005

Multiple roles of Rbx1 in the VBC-Cul2 ubiquitin ligase complex.

The importance of the ubiquitin system largely depends on ubiquitin ligases, E3s, as they determine the specificity of the system. Rbx1/ROC1/Hrt1, a RING finger protein, functions as an important component of the cullin-containing SCF and VBC-Cul2 ligases. Modification of cullins by NEDD8 (NEDDylation), has been shown to be essential for the E3 activity of both SCF and VBC-Cul2, and it was suggested that Rbx1 acts as the E3 for cullin NEDDylation. RING finger is composed of eight cysteine and histidine residues that bind to zinc ions. Rbx1 is a highly evolutionarily conserved protein; however, the eighth coordination residue in its RING finger is aspartate (D97) rather than cysteine. Substitution of D97 with each of the other 19 amino acids demonstrates that aspartate is superior to cysteine in cullin NEDDylation. Interestingly, however, different D97 mutants demonstrate different activities towards 6 cullins tested. Importantly, we were able to discriminate between the NEDDylating activity of Rbx1 and its involvement in the ubiquitylation reaction within the context of VBC-Cul2. Moreover, while Rbx1 is not involved in governing the stability of SCF, Rbx1 mutants destabilize VBC-Cul2. Taken together, these results indicate that various mechanisms regulate both the activities and the stability of cullin-based ligases.

Carrier Proteins↗